DEAD-box proteins (DBPs) are key regulators of RNA structure inside the cell and are generally considered to function as ATP-dependent RNA helicases. Although DBPs are implicated in a wide range of cellular processes, the mechanisms by which these factors function inside the cell are not well understood. The objective of the proposed work is elucidate and characterize the in vivo functions of two DBPs from E. coli, DeaD and SrmB. Published data and preliminary results from our laboratory indicate that both factors participate in multiple, partially overlapping aspects of RNA metabolism in the cell. To define their cellular functions in detail, we propose three specific aims. First, both SrmB and DeaD have been implicated in ribosome assembly. Through the identification of genetic suppressors that alleviate the cellular defects caused by an absence of SrmB, we have identified the ribosomal role of this protein is to stimulate the production of a key ribosomal protein, L13. The experiments proposed in this aim will test the hypothesis that SrmB regulates the structure of the mRNA that encodes L13, and thereby, affects L13 production. We will also analyze genetic suppressors of cells that lack DeaD to elucidate the mechanism by which this DBP regulates ribosome assembly. In the second aim, we will globally analyze the regulation of mRNAs in E. coli. Our preliminary data indicate that an absence of either DBP affects the abundance of hundreds of mRNAs. Studies will be performed to test the hypothesis that DBP-mediated structural changes affect RNA expression at the level of transcription termination, stability or translation. We will also investigate whether ATPase-independent activities of the DBPs, such as the ones we have recently identified, are important for transcript regulation in the cell. In the third aim, we will apply a validated cross-linking approach to identify the binding sies of the DBPs on mRNAs and determine RNA binding motifs. Elucidating the basis for DBP- mediated recognition of RNA is critical to understand the underlying basis for RNA regulation. Collectively, these studies will provide multiple insights into the functions of this important famly of RNA regulators, the basis for their interaction with RNA and the mechanisms through which RNA substrates are regulated.